CN103642936A - Primer probe composition and kit for specific detection of 55 type adenovirus nucleic acid - Google Patents

Primer probe composition and kit for specific detection of 55 type adenovirus nucleic acid Download PDF

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CN103642936A
CN103642936A CN201310535934.0A CN201310535934A CN103642936A CN 103642936 A CN103642936 A CN 103642936A CN 201310535934 A CN201310535934 A CN 201310535934A CN 103642936 A CN103642936 A CN 103642936A
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primer
test kit
pcr reaction
specific detection
probe
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CN103642936B (en
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丁小静
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JIANGSU UNINOVO BIOLOGICAL TECHNOLOGY Co Ltd
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Abstract

The invention relates to an oligonucleotide sequence composition for specific detection of 55 type adenovirus nucleic acid in sample using fluorescence PCR technology as well as a kit containing the composition. The kit can sensitively detect and identify 55 type adenovirus nucleic acid existed in sample, and the detection lower limit is 20 copies per reaction system, and has important application values in disease monitoring, clinic diagnosis and other fields.

Description

Primer probe combinations and test kit for the specific detection of 55 type adenovirus
Technical field
The present invention relates to a kind of employing Fluorescence PCR assay, the oligonucleotide sequence combination that in specific detection sample, 55 type adenoviral nucleic acids exist, and the test kit that comprises this combination.
Background technology
Adenovirus is a kind of double-stranded DNA virus without coating, wire, to respiratory tract, gi tract, urethra and bladder, eye, liver etc., all can infect.
The respiratory tract infection epidemic outbreaks that adenovirus causes has report at home and abroad time, mainly betides the crowd in enclosed environment, as boarding school, and Kindergartens.Clinical symptom, except heating, shiver with cold, headache, cough, often has obvious pharyngalgia and bottleneck throat ulcer; Clinically, adenovirus respiratory tract infection is divided into following several types: Clinical types in febris acuta pharyngitis, pharyngoconjunctivitis, acute bronchitis and pneumonia 4.
At present, adenovirus has 55 types, adenovirus 4,7 bezonians of He21Xing Shi army and other crowds in enclosed environment, the common disease substance breaking out as boarding school, Kindergartens acute respiratory disease, 55 type adenovirus are found in China, be the new virus that adenovirus type XI and 15 type gene recombination form, once in 2006, in northern China school student colony, caused respiratory tract epidemic outbreaks, take light-duty case as main.
In laboratory is detected, PCR method will substitute because its fast and convenient feature presents gradually that to take that microbial culture and serology detect be the trend of main traditional detection method.And for PCR method, the specificity of primer is the basis of specificity and the susceptibility of its detection.The present invention is directed to 55 type adenovirus special target sequence design primer and Taqman probe, utilize the method for real-time PCR, be used for differentiating and detect 55 type adenoviral nucleic acids in sample, can obtain fast specific detection result.
Summary of the invention
In order to detect more exactly sample 55 type adenoviral nucleic acids, exist, the invention provides the oligonucleotide sequence combination of 55 type adenovirus in a kind of specific detection sample and the test kit that comprises this combination, it is characterized in that in the PCR reaction solution of combined sequence or test kit, the primer for nucleic acid amplification is:
P1：5`-CAGGATGCTTCGGAGTACCT-3`,
P2：5`-CTTATTTCCCAGATTGAAGTAGGT-3`。
P1 and P2 are for for 55 type adenoviral gene group-specific sequences Design the Auele Specific Primer that filters out through preliminary experiment.
Feature of the present invention is also, the oligonucleotide probe for fluorescent signal monitoring in the PCR reaction solution of combined sequence or test kit is:
Probe1：5`-X 1-CCGGGTCTGGTGCAGTTTGCC-Y 1-3`。
X 1for fluorescence report group, Y 1for fluorescent quenching group.Probe1 is for for 55 type adenoviral gene group-specific sequences Design the specific probe that filters out through preliminary experiment.
Feature of the present invention is also, test kit comprises PCR reaction solution, enzyme mixation, negative quality control product and positive quality control product.Wherein PCR reaction solution mainly contains above-mentioned primer and probe, reaction buffer, Mg 2+with dNTP etc., enzyme mixation mainly contains warm start Taq enzyme, and negative quality control product is deionized water, and positive quality control product is to contain the nucleic acid that detects target sequence.
A preferred embodiment of the present invention is: the fluorophor of the oligonucleotide probe of monitoring for fluorescent signal in the PCR reaction solution of combined sequence or test kit divides, wherein Probe1 fluorescence report radicals X 1for Fam, fluorescent quenching group Y 1for Eclipse.
Another preferred embodiment of the present invention is: the PCR reaction solution of test kit comprises above-mentioned Auele Specific Primer and specific probe, belongs to real-time fluorescence substance PCR and detects, and can in same reaction system, to 100 type adenoviral nucleic acids, detect.
Another preferred embodiment of the present invention is: the PCR reaction cycle parameter of test kit is 94 ℃, 2min; Enter the cycle stage: 94 ℃ of sex change 10s, 56 ℃ of annealing 50s, 72 ℃ are extended 15s, 40 circulations of coreaction.
Another preferred embodiment of the present invention, the PCR reaction system that test kit is selected is 20 μ l, comprise 2 * PCR damping fluid, 10 μ l, 10mmol/L dNTP 1.0 μ l, each 0.5 μ l of 25 μ mol/L primers, 10 μ mol/L probe 0.2 μ l, warm start Taq enzyme mixation 0.4 μ l, sample DNA 2 μ l, adding aqua sterilisa to end-body is 20 μ l.
Test kit provided by the invention is to being limited to 20 every reaction systems of copy under the detection of 55 type adenoviral nucleic acids.
The present invention designs respectively Auele Specific Primer and probe according to 55 type adenoviral gene group conserved regions respectively, the test kit providing can detect the nucleic acid of 55 type adenovirus, but can not detect the nucleic acid of non-55 type adenovirus pathogenic agent, illustrate that test kit has good specificity.
Test kit provided by the invention can complete detection in 2 hours, and the disease surveillance and the clinical diagnosis that can be 55 type adenovirus provide experimental basis.
Accompanying drawing explanation
Fig. 1 is that substance real-time fluorescence PCR detects 55 type adenovirusthe amplification curve diagram of nucleic acid sensitivity.5 curves are from left to right respectively 55 type adenovirus specific PCR fragments and build plasmid gradient dilution (10 5-10 1) rear amplification.X-coordinate is reaction cycle number, and ordinate zou is the DRn value of different cycle number fluoroscopic examination signals.
Fig. 2 is that real-time fluorescence substance PCR detection system is to 55 type adenoviral nucleic acid detection specificity amplification curve diagrams.There is S type amplification curve in 55 type adenovirus, and S type amplification curve does not all appear in other pathogenic micro-organism Quality Control bacterium.X-coordinate is reaction cycle number, and ordinate zou is the DRn value of different cycle number fluoroscopic examination signals.
Embodiment
Below in conjunction with specific embodiment, describe the preferred embodiment of the present invention in detail.It is pointed out that the embodiment listing is only the object of exemplary illustration here, and should be interpreted as any restriction to the scope of the invention.The reagent such as use therein test kit, damping fluid are only the concrete reagent of selecting in this specific embodiment, should be understood that those skilled in the art can select the corresponding reagent of other companies to realize object of the present invention as required.
1, the design of primer and TaqMan probe is with synthetic
Utilize Blast instrument to analyze the 55 all type adenoviral gene group sequences in Genbank and domestic and foreign literature, select respectively its stable conservative region as detecting target sequence, and survey target sequence design and synthetic primer and probe (in Table 1) for this.Primer and probe are synthetic by the precious biotech firm in Japanese TaKaRa Dalian, and wherein the detection probes 5 ' of 55 type adenovirus is held flag F AM fluorophor, 3 ' end mark Eclipse fluorescent quenching group.
2, detect the preparation of bacterial classification
The 55 type adenovirus of using in the present embodiment and other negative control bacterial strains (adenovirus type III, 7 types, influenza virus A H1N1, AH3N2, streptococcus pneumoniae, b type hemophilus influenzae, mycoplasma pneumoniae, Chlamydia pneumoniae and moraxelle catarrhalis) are all bought in Nat'l Pharmaceutical & Biological Products Control Institute.
3, the extracting of bacterial strain DNA
Select the QIAamp DNA Mini Kit(of Qiagen company article No.: 51306) extract bacterial strain DNA.Concrete steps test kit is with reference to process specifications.
4, the screening of primer and probe
Adopt primer and probe difference 55 type adenovirus of Detection and Extraction and the genomic dna of negative control bacterial strain bacterium of design, through experiment repeatedly, filter out sensitivity, specificity and the best primer probe combinations (seeing sequence table, 55 type adenovirus forward primer p1, reverse primer p2 and probe probe1) of repeatability.
5, the structure of standard substance and preparation
Utilize respectively p1, p2 primer, pcr amplification 55 type adenovirus specific gene fragments, by PCR product cloning to pMD-18T carrier, transform DH5a intestinal bacteria, utilize alkaline lysis method of extracting positive colony plasmid, utilize ultraviolet-visible pectrophotometer to be determined at respectively the light absorption ratio of wavelength 260nm place, the DNA of 280nm place, then calculate plasmid concentration and purity.Then 10 times of gradient dilutions to 10 copy every microlitres.
6, reaction condition optimization
The key elements such as primer, probe, enzyme are optimized one by one, definite reaction system is: 2 * PCR damping fluid, 10 μ l, 10mmol/L dNTP 1.0 μ l, 25 μ mol/L primers each 0.5 μ l, 10 μ mol/L probe 0.2 μ l, Takara polysaccharase mixture 0.4 μ l, template 2ul, adding aqua sterilisa to end-body is 20 μ l.
According to amplified fragments length, primer and the annealing temperature of probe and the characteristic of enzyme, mainly reaction annealing temperature and extension time are optimized, final definite loop parameter is: 94 ℃, 2min; Enter the cycle stage: 94 ℃ of sex change 10s, 56 ℃ of annealing 50s, 72 ℃ are extended 15s, 40 circulations, each circulates in annealing stage and gathers fluorescent signal.
After finishing, amplification by identical conditions analytical data, determines the Ct value of each sample.
7, the evaluation of detectability
The detectability that the test kit providing of the present invention is provided with the positive criteria product in above-mentioned 5, positive criteria product concentration is: 1 * 10 5copies/ μ l, 1 * 10 4copies/ μ l, 1 * 10 3copies/ μ l, 1 * 10 2copies/ μ l, 1 * 10 5copies/ μ l, 1 * 10 5copies/ μ l, test kit provided by the invention is to being limited to 20 every reaction systems of copy under the detection of 55 type adenoviral nucleic acids.
8, the evaluation of detection specificity
The bacterial strain DNA of take in above-mentioned 2 has evaluated the specificity of this test kit as template.Equal visible clear and definite amplification curve when 55 type adenovirus DNAs are detected, when above-mentioned 9 kinds of other pharyngeal encountered pathogenic microbial DNAs are detected, do not produce positive amplification curve, illustrate between probe that we use and primer and other bacterial strains that we select and do not have cross reaction.
Although above in a preferred manner; by specific embodiment exemplary illustration some embodiment of the present invention; but it will be understood by a person skilled in the art that; the present invention is not limited to disclosed embodiment above; but can to it, modify according to the knowledge of the technical field of the invention, institute makes an amendment and can not exceed the scope of protection of present invention.For example, fluorescent real time PCR used in the present invention also can adopt fluorophor and the fluorescent quenching group mark substance in addition of pointing out in embodiment listed in specification sheets as required, as markers such as Tet, FAM, HEX, TAMRA, ROX, Cy3, TxRd, JOE; Or other mark systems outside use Taqman technology, fluorescent probe labeling techniques such as MGB probe, molecular beacon MB probe, scorpion shape probe, fluorescence double cross probe; Or use the chimeric method of dyestuff as saturable dyes such as the unsaturated dyestuffs such as SYBR Green I and LC Green, as long as it has used specific primer sequence of the present invention, get final product the existence of qualitative or detection by quantitative goal gene, and then detect specifically the existence of 55 type adenovirus.So the change that these those skilled in the art understand and the replacement of customary means also fall within the scope of protection of the present invention.Protection scope of the present invention should be limited by appending claims.
He Chuan bio tech ltd, <110> Jiangsu
Primer probe combinations and test kit for the specific detection of <120> 55 type adenovirus
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<220>
<221> primer_bind
<222> (1)..(20)
<223> is according to the primer sequence of genome conserved regions design, for nucleic acid amplification and detection.
<400> 1
caggatgcttcggagtacct 20
<210> 2
<211> 24
<212> DNA
<213> artificial sequence
<220>
<221> primer_bind
<222> (1)..(24)
<223> is according to the primer sequence of genome conserved regions design, for nucleic acid amplification and detection.
<400> 2
cttatttcccagattgaagtaggt 24
<210> 3
<211> 21
<212> DNA
<213> artificial sequence
<220>
<221> misc_binding
<222> (1)..(21)
<223> is according to the probe sequence of genome conserved regions design, for nucleic acid amplification and detection.
<400> 3
ccgggtctggtgcagtttgcc 21

Claims (9)

1. for a combination of primers for specific detection sample 55 type adenoviral nucleic acids, this cover combined sequence is characterised in that and comprises following primer:
P1：5`-CAGGATGCTTCGGAGTACCT-3`,
P2：5`-CTTATTTCCCAGATTGAAGTAGGT-3`。
2. the primer for specific detection sample 55 type adenoviral nucleic acids and oligonucleotide probe combination, wherein primer is primer claimed in claim 1, oligonucleotide probe is:
Probe1：5`-X 1-CCGGGTCTGGTGCAGTTTGCC-Y 1-3`；
X 1for fluorescence report group, Y 1for fluorescent quenching group.
3. as claimed in claim 2, this cover combined sequence feature is also, wherein Probe1 fluorescence report radicals X 1for Fam, fluorescent quenching group Y 1for Eclipse.
4. as claimed in claim 2, this cover combined sequence feature is also, can be used for substance real-time fluorescence polymerase chain reaction and detects, and a reaction system can detect 55 type adenoviral nucleic acids.
5. for the test kits of specific detection sample 55 type adenoviral nucleic acids, comprising PCR reaction solution, enzyme mixation, negative quality control product and positive quality control product,
It is characterized in that in PCR reaction solution, the primer sequence for nucleic acid amplification reaction is as follows:
P1：5`-CAGGATGCTTCGGAGTACCT-3`,
P2：5`-CTTATTTCCCAGATTGAAGTAGGT-3`。
6. test kit as claimed in claim 5, is characterized in that in PCR reaction solution for the sequence of the oligonucleotide probe of fluorescent signal monitoring as follows:
Probe1：5`-X 1-CCGGGTCTGGTGCAGTTTGCC-Y 1-3`，
X 1for fluorescence report group, Y 1for fluorescent quenching group.
7. test kit as claimed in claim 5, is further characterized in that in PCR reaction solution the oligonucleotide probe for fluorescent signal monitoring: Probe1 fluorescence report radicals X wherein 1for Fam, fluorescent quenching group Y 1for Eclipse.
8. test kit as claimed in claim 5, be further characterized in that PCR reaction system is 20 μ l, comprise 2 * PCR damping fluid, 10 μ l, 10mmol/L dNTP 1.0 μ l, each 0.5 μ l of 25 μ mol/L primers, 10 μ mol/L probe 0.2 μ l, warm start Taq enzyme mixation 0.4 μ l, sample DNA 2 μ l, adding aqua sterilisa to end-body is 20 μ l.
9. test kit as claimed in claim 5, is further characterized in that PCR reaction cycle parameter is:
94 ℃, 2min; Enter the cycle stage: 94 ℃ of sex change 10s, 56 ℃ of annealing 50s, 72 ℃ are extended 15s, 40 circulations of coreaction.
CN201310535934.0A 2013-11-04 2013-11-04 The specific detection of 55 type adenoviruss primed probe combination and test kit Active CN103642936B (en)

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CN105861751A (en) * 2016-05-16 2016-08-17 浙江省医学科学院 Primer pair and fluorescent quantitative PCR kit for detecting mouse adenovirus and application thereof
WO2016165340A1 (en) * 2015-04-15 2016-10-20 广州福宸生物技术有限公司 Human type 55 replication defective adenovirus vector, method for preparing same and uses thereof
CN108004348A (en) * 2017-09-27 2018-05-08 湖北朗德医疗科技有限公司 A kind of primer, probe and kit for being used to detect enteric adenovirus
CN109266787A (en) * 2018-11-29 2019-01-25 四川出入境检验检疫局检验检疫技术中心 A kind of Nucleic acid combinations, kit and application and detection method detecting 5 type cow adenovirus
CN109402067A (en) * 2018-10-18 2019-03-01 北京晨寰生物科技有限公司 One plant of 55 type adenovirus and its vaccine product of preparation
CN109504802A (en) * 2018-11-22 2019-03-22 李越希 A kind of RPA method, its primer special and probe and purposes detecting 11/55 type adenovirus hominis
CN109988869A (en) * 2019-04-23 2019-07-09 深圳市亚辉龙生物科技股份有限公司 Nucleic acid compositions, detection unit, micro-fluidic chip and detection device
CN110055354A (en) * 2019-04-23 2019-07-26 深圳市亚辉龙生物科技股份有限公司 Nucleic acid compositions, detection unit, micro-fluidic chip and detection device
CN110106285A (en) * 2019-03-19 2019-08-09 中国疾病预防控制中心病毒病预防控制所 A kind of dual isothermal nucleic acid amplification method containing internal reference of 3 type adenovirus hominis of quick detection
CN113528709A (en) * 2021-09-13 2021-10-22 北京华瑞康源生物科技发展有限公司 Fluorescent quantitative PCR detection method capable of covering 13-type adenovirus and kit

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WO2016165340A1 (en) * 2015-04-15 2016-10-20 广州福宸生物技术有限公司 Human type 55 replication defective adenovirus vector, method for preparing same and uses thereof
CN105861751A (en) * 2016-05-16 2016-08-17 浙江省医学科学院 Primer pair and fluorescent quantitative PCR kit for detecting mouse adenovirus and application thereof
CN105861751B (en) * 2016-05-16 2019-04-09 浙江省医学科学院 A kind of primer pair for detecting mouse adenovirus, PCR kit for fluorescence quantitative and its application
CN108004348A (en) * 2017-09-27 2018-05-08 湖北朗德医疗科技有限公司 A kind of primer, probe and kit for being used to detect enteric adenovirus
CN109402067A (en) * 2018-10-18 2019-03-01 北京晨寰生物科技有限公司 One plant of 55 type adenovirus and its vaccine product of preparation
CN109402067B (en) * 2018-10-18 2022-08-09 国科丹蓝投资(北京)有限公司 55-type adenovirus and vaccine product prepared from same
CN109504802A (en) * 2018-11-22 2019-03-22 李越希 A kind of RPA method, its primer special and probe and purposes detecting 11/55 type adenovirus hominis
CN109266787A (en) * 2018-11-29 2019-01-25 四川出入境检验检疫局检验检疫技术中心 A kind of Nucleic acid combinations, kit and application and detection method detecting 5 type cow adenovirus
CN110106285B (en) * 2019-03-19 2022-06-03 中国疾病预防控制中心病毒病预防控制所 Internal reference-containing dual isothermal nucleic acid amplification method for rapidly detecting 3-type human adenovirus
CN110106285A (en) * 2019-03-19 2019-08-09 中国疾病预防控制中心病毒病预防控制所 A kind of dual isothermal nucleic acid amplification method containing internal reference of 3 type adenovirus hominis of quick detection
CN110055354A (en) * 2019-04-23 2019-07-26 深圳市亚辉龙生物科技股份有限公司 Nucleic acid compositions, detection unit, micro-fluidic chip and detection device
CN109988869A (en) * 2019-04-23 2019-07-09 深圳市亚辉龙生物科技股份有限公司 Nucleic acid compositions, detection unit, micro-fluidic chip and detection device
CN109988869B (en) * 2019-04-23 2023-08-11 深圳市亚辉龙生物科技股份有限公司 Nucleic acid composition, detection unit, microfluidic chip and detection device
CN110055354B (en) * 2019-04-23 2023-11-03 深圳市亚辉龙生物科技股份有限公司 Nucleic acid composition, detection unit, microfluidic chip and detection device
CN113528709A (en) * 2021-09-13 2021-10-22 北京华瑞康源生物科技发展有限公司 Fluorescent quantitative PCR detection method capable of covering 13-type adenovirus and kit
CN113528709B (en) * 2021-09-13 2021-12-10 北京华瑞康源生物科技发展有限公司 Fluorescent quantitative PCR detection method capable of covering 13-type adenovirus and kit

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Denomination of invention: Primer probe combination and kit for specific detection of adenovirus type 55

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